Chemodenervation refers to the use of an agent to prevent a nerve from stimulating its target tissue, e.g. a muscle, a gland or another nerve. Chemodenervation is for example performed with phenol, ethyl alcohol, or botulinum toxin. Chemodenervation is for example appropriate in patients with localized spasticity in one or two large muscles or several small muscles. It may be used to alleviate symptoms such as muscle spasm and pain, and hyperreflexia.
Chemodenervating agents block neuromuscular transmission at the neuromuscular junction, causing paralysis or paresis of the affected skeletal muscles. The term “paresis” is defined hereinunder as a condition typified by partial loss of movement, or impaired movement. This is accomplished either by acting presynaptically via the inhibition of acetylcholine (ACh) synthesis or release, or by acting postsynaptically at the acetylcholine receptor. Example of drugs that act presynaptically are botulinum toxin, tetrodotoxin and tetanus toxin.
The term “chemodenervation” also encompasses all effects which directly or indirectly are induced by the chemodenervating agent, therefore also comprising upstream, downstream or long-term effects of said chemodenervating agent. Therefore, presynaptic effects are also encompassed as well as postsynaptic effects; tissue effects and/or indirect effects via spinal or afferent neurons.
One chemodenervating agent, botulinum toxin, although being one of the most toxic compounds known to date, has in the past been used for the treatment of a large number of conditions and disorders, some of which are described in e.g. PCT/EP 2007/005754. Furthermore, commercial forms of botulinum toxin type A based on the botulinum toxin A protein complex are available under the tradename BOTOX® (Clostridium botulinum toxin type A purified toxin complex, (900 kDa), Allergan Inc.) and under the tradename DYSPORT® (Clostridium botulinum type A toxin-haemagglutinin complex; lpsen Ltd.), respectively. A pharmaceutical composition based on a higher purified toxin preparation and comprising the neurotoxic component of botulinum toxin type A free of complexing proteins in isolated form is commercially available in Germany from Merz Pharmaceuticals GmbH under the tradename XEOMIN® (incobotulinumtoxinA; Clostridium botulinum type A neurotoxin (150 kDa), free of Complexing proteins).
The anaerobic, Gram-positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. Botulinum toxin A (BoNT/A) is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) serotype A is a LD50 in mice. However, despite its toxic effects, botulinum toxin complex as well as the pure neurotoxin have been used as a therapeutic agent in a large number of diseases.
Botulinum toxins are released from lysed Clostridium cultures generally in the form of a protein responsible for the toxic properties of the botulinum toxin (the neurotoxic component) in association with other bacterial proteins (the non-toxic “complexing proteins” or “clostridial proteins”), which together form a toxin complex also designated “botulinum toxin complex”. The botulinum toxin complex is metastable in nature, since its stability appears to depend on various factors such as e.g. salt concentration and/or pH. The molecular weight of the complex may vary from about 300,000 to about 900,000 Da i.e. from 300 kDa to about 900 kDa. The complexing proteins are, for example, various hemagglutinins. The proteins of this toxin complex are not toxic themselves but are believed to provide stability to the neurotoxic component and are responsible for oral toxicity in Botulinum intoxications. There are seven antigenically distinct serotypes of botulinum toxin, namely botulinum toxin A, B, C1, D, E, F and G. Wherever the botulinum toxin sero-type A, B, C1, D, E, F or G are mentioned, also known variants of the sero-types are encompassed, like serotypes A1, A2, A3, A4 etc.
The component of clostridial toxins responsible for its high toxicity is the neurotoxic component or protein (Mw≈150 kD, exact molecular weight depending of the serotype). The several different serotypes differ in their amino acid sequence, but possess all a similar structure: a light chain (LC) of approximately 50 kDa and a heavy chain (HC) of approximately. 100 kDa, which may be linked by one or more disulfide bonds (for a review see e.g. Simpson L L, Ann Rev Pharmacol Toxicol. 2004; 44:167-93). The neurotoxic component of the botulinum toxin complex is initially formed as a single poly-peptide chain. In the case of serotype A, for example, proteolytic processing of the polypeptide results in an activated polypeptide in the form of a dichain polypeptide consisting of a heavy chain and a light chain, which are linked by a disulfide bond. In humans, the heavy chain mediates binding to pre-synaptic cholinergic nerve terminals and internalization of the toxin into the cell. The light chain is believed to be responsible for the toxic effects, acting as zinc-endopeptidase and cleaving specific proteins responsible for membrane fusion (SNARE complex) (see e.g. Montecucco C., Shiavo G., Rosetto O: The mechanism of action of tetanus and Botulinum neurotoxins. Arch Toxicol 1996; 18 (Suppl.): 342-354)).
The term “botulinum toxin” as used throughout the present application, refers to the neurotoxic component devoid of any other clostridial proteins, but also to the “botulinum toxin complex”. The term “botulinum toxin” is used herein in cases when no discrimination between the toxin complex and the neurotoxic component is necessary or desired. “BoNT” or “NT” are common used abbreviations for botulinum neurotoxin or neurotoxin, respectively. The neurotoxic subunit of the botulinum toxin complex is referred in this document as the “neurotoxic component” or the“neurotoxic component free of complexing proteins”. The production of the neurotoxic component of botulinum toxin type A and B are described, for example, in the international patent application WO 00/74703.
The several serotypes differ by their duration of therapeutic effect: The normal period of activity of botulinum toxin A drugs is, if injected intramuscular in humans, between 3 and 4 months. In single cases the period can even extend to more than 12 months. During the treatment of sweat glands, an activity of even 27 months has been reported (Bushara K., botulinum toxin and rhinorrhea, Otolaryngol. Head. Neck. Surg., 1996; 114(3):507 and The Laryngoscope 109: 1344 1346:1999). The period of activity for botulinum toxin type C1 is comparable with the period of activity of botulinum toxin A (Eleopra et al., 1997 & 2002). Surprisingly the period of action is much shorter in rodents (e.g. mice) as compared to humans: Approximately 1-2 months for botulinum toxin A, 21 days for botulinum toxin B and only 4 days for botulinum toxin E (DePaiva et al., 1999, Juradinski et al., 2001).
Foran et al. analyzed in 2003 the time period of action in vitro on cerebellum-neurons of rats and found half-times of the inhibition of glutamate exocytosis for botulinum toxin A of more than 31 days; for botulinum toxin type C1 of more than 25 days; for botulinum toxin type B of approximately 10 days; for botulinum toxin type F of approximately 2 days and for botulinum toxin type E of only 0.8 days.
The time period of activity of botulinum toxin type A during e.g. the treatment of dystonias (e.g. Torticollis, Blepharospasmus) in humans is between 3 to 4 months. After this period the patient has to receive another injection of a botulinum toxin-containing drug. It would be of great advantage for the patient to prolong the time period of action of the neurotoxin. In doing so, the number of necessary injections per year would be reduced as well as the overall amount of clostridial proteins. This again would reduce the risk of the production of antibodies against the foreign protein. Therefore, the provision of a botulinum toxin with prolonged persistency would be desirable.
However, not always long-term paralyzation is desired. For example in certain cosmetical treatments sometimes only temporal “fine-adjustments” are required. To achieve a reduction of persistency the physician was restricted by the prior art methods to either the reduction of volume or the switch of serotype. These techniques proved to result in unsatisfying results and required profound knowledge both of the activity kinetics as well as the antigenicity of the different neurotoxin serotypes. Therefore, the provision of a neurotoxin with a “built-in” adjustment of persistency would be a major improvement.
US 2003/0219462, EP1849801 and WO 02/08268. disclose modified Botulinum toxins with added leucine- or tyrosine-based motifs to the native neurotoxin.
The idea for these alterations is based on the observation that certain leucine- or tyrosine-based motifs enable the localization of the light chain of the neurotoxic component of certain subtypes to the inner membrane of the target cell. This mechanism was hypothesized to change the persistency of certain light chains. Until now, however, the authors failed to provide any evidence for such an effect and newer experiments suggest that the whole hypothesis is inaccurate.
Furthermore, even if in certain cases an addition of motifs would lead to a membrane localization, such an approach is not applicable to modifications of Botulinum toxin A. This is because the native light chain of Botulinum toxin type A is already localized to the inner cell-membrane, therefore an additional tethering to the membrane does not provide any additional benefit.
Therefore the present invention followed a different path. As it has been found as disclosed in this application, the addition of a second light chain to the neurotoxin, which still possesses its proteolytic activity, leads to an alteration of the time period of activity. Depending on the combination of serotypes used, the time-period can be prolonged, allowing for the production of custom-tailored neurotoxins. It is envisaged to provide the physician with a range of neurotoxins, whose serotype is independent of their persistency, allowing for a more standardized treatment.